Techniques for assaying a sample for the presence and/or concentration of specific substances are known to those skilled in the art. Examples of such techniques include nucleic acid hybridization assays, various protein-binding methodologies including radioimmunoassay, enzyme immunoassay, enzyme-linked immunoassays. All of these techniques involve the binding of a compound to some sort of specific receptor and accordingly fall into the general category of ligand/anti-ligand assays, a category not limited to any special type of interaction occurring in the assay or to any particular type of components participating in the reaction.
All ligand/anti-ligand assays are based on two premises: (1) that certain pairs of substances (the ligand and the anti-ligand) have a strong and specific affinity for each other, that is, they will tend to bind to each other, while binding little or not at all to other substances; and (2) that methods and devices can be developed that allow detection of ligand/anti-ligand binding interactions once complexes have formed. As used herein, ligand is defined as the substance to be detected, and anti-ligand as the substance used to probe for the presence of the ligand.
Ligand/anti-ligand reactions can be detected by a variety of methods, using various markers to label either the ligand or anti-ligand to permit detection of the reaction product. Currently, the most commonly used markers include enzymes and fluorochromes and radioactive compounds. Immobilization of either the ligand or anti-ligand will facilitate detection in many cases.
Ligand/anti-ligand assays can be generally classed into two categories, heterogeneous and homogeneous. Heterogeneous assays require separation of the bound-labeled component from the free-labeled component prior to detection of the reaction product. Homogeneous assays do not require such a separation step. These assays can further be (1) competitive, for example, where ligand competes for labeled anti-ligand with a solid-phase ligand or where anti-ligand competes with labeled anti-ligand for a solid-phase ligand or (2) noncompetitive where there is a direct relationship between label and ligand or anti-ligand.
Ligand/anti-ligand assay methods can be applied to fluorescent immunoassay, enzyme immunoassay and radioimmunoassay techniques to detect the presence or absence of antibodies or antigens, i.e., the ligand, in a sample. In recent years the use of enzyme immunoassays (EIA) and enzyme-linked immunoassays (ELISA) has become increasingly important for the qualitative and semi-quantitative detection of a wide variety of substances. In ELISA methodology, antigens can be labeled directly or indirectly by use of enzyme-labeled antibodies which, under appropriate conditions, catalyze a reaction with a substrate. The enzyme activity is detected by formation of a colored reaction product i.e., a colored end point that may be easily detected by eye or measured by spectroscopic or reflectance means. Several enzymes, including alkaline phosphatase, horseradish peroxidase (HRP) and glucose oxidase, have been coupled to both antigen and antibody. HRP is commonly used and several substrates are available for it. For visual detection in an HRP assay, the substrate will usually comprise a solution of a peroxide such as hydrogen peroxide and a chromogenic material such as o-phenylenediamine or tetramethylbenzidine which manifests a color upon oxidation.
In fluorescent immunoassay techniques antigens can be labeled either directly or indirectly with fluorochrome-labeled antibodies. Fluorochromes are dyes that absorb radiation (e.g., ultraviolet light), are excited by it, and emit light (e.g., visible light). The most commonly used fluorochromes are fluorescein isothiocyante and tetramethylrhodamine isothiocyanate.
Ligand/anti-ligand assays also include protein binding assays wherein a specific binding protein is used as an anti-ligand to probe a sample for the protein which it binds. The reaction product of such protein-binding assays, can also be detected using radioactive, fluorescent or enzyme labels. Either the binding protein or its target protein may be labeled.
Yet another ligand/anti-ligand assay that is becoming increasingly important is the nucleic acid hybridization assay, e.g., the DNA probe assay, which uses a "probe" strand of nucleic acid as an anti-ligand to test for the presence of a complementary DNA sequence. DNA probe assays, like immunoassays, often use radioactive labels, fluorescent labels or enzyme labels. Both immunoassays and DNA probe assays have used luminescent labels as well.
In many cases, ligand/anti-ligand techniques require large sample volumes, are time consuming and involve multi-step procedures. For these reasons, it is desirable to carry out such assays with the aid of a device which facilitates the reaction between ligand and anti-ligand, for example, by using a minimum amount of sample, requiring fewer steps, and enabling the reaction to take place rapidly, within the device without transfer of the ligand being assayed. It is also desirable to carry out such assays with the aid of a device which facilitates detection of the reaction product, in some cases, by eliminating the need for detection equipment or by providing the reaction product in a form which is detectable by equipment without further processing of the reaction product.
Immobilization of either the ligand or anti-ligand will facilitate detection in many ligand/anti-ligand assays. Useful ligand/anti-ligand assay systems for assaying drugs, hormones, peptides, proteins, enzymes, nucleic acids, antibodies, haptens, antibiotics, viri, infectious agents, tumor markers and the like commonly are solid phase systems using ligands or anti-ligands immobilized on a water-insoluble carrier such as metal, glass, or plastic.
U.S. Pat. No. 4,090,849 provides a diagnostic device for the detection of biological particles wherein a metal sheet is used as the solid phase upon which a layer of protein is applied.
U.S. Pat. No. 4,280,992 provides immunologically active substance-frosted glass conjugates for use in assays of physiologically active substances using the same. The glass solid phase of '992 is frosted by physical means, e.g., sandblasting, or by chemical treatment, e.g., etching. '992 teaches use of these glass conjugates in the form of frosted tubes and beads.
The ability of proteins to absorb to plastic materials is a well-known phenomenon to those skilled in the art, and various ligand/anti-ligand assays have been developed in which a protein is immobilized on plastic.
U.S. Pat. No. 4,197,361 discloses an immunoassay for the sandwich technique in which antibody (or antigen) is bound to a plastic, and after reaction with a test sample and then fluorescently tagged antibody (or antigen), fluorescense is read directly from the strip in a fluorometer. '361 further discloses sandblasting the plastic strip to increase the surface area.
Assays have also been developed wherein groups reactive with a particular type of ligand or anti-ligand are grafted to the surface of the plastic. U.S. Pat. No. 4,317,810 discloses a water insoluble polymeric matrix which has a layer of reactive groups grafted onto its opposing surfaces, wherein the surfaces have a designed configuration in the form of a plurality of ridges and depressions so that when the matrix is placed in a vial containing solution both surfaces will be substantially in complete contact with the solution and there will be a minimum of surface-to-surface contact between the matrix and the bottom of the vial.
Although presently available test devices have provided means to increase the sensitivity and ease of carrying out ligand/anti-ligand assays, more sensitive and easier assays are needed. Thus, alternative devices are being sought which require, for example, smaller amounts of sample and fewer steps, and which provide a more convenient methodology.